Identification of regions of constitutive heterochromatin and sites of ribosomal DNA ( rDNA ) in Rhogeessa hussoni ( Genoways & Baker , 1996 ) ( Chiroptera ; Mammalia ; Vespertilionidae )

There is scarce information about the geographical distribution, biological and cytogenetic data from the Rhogeessa hussoni. This study aims to characterize its chromosome composition through chromosome bandings to visualize regions of constitutive heterochromatin (CGB bands) and sites of ribosomal DNA (rDNA) in R. hussoni’s karyotype. A female specimen of R. hussoni was collected in the “Parque Municipal Mário Viana” Conservation Unit, Nova Xavantina, Mato Grosso, CentralWest region of Brazil. The karyotype constitution was 2n=52 and NF=54. The CBG bands evidenced a sex X chromosome nearly completely constituted by heterochromatin. The Rhogeessa hussoni has two sites of rDNA located in a single pair (pair 25) of autosomal chromosomes. We carried out the first cytogenetic characterization of R. hussoni, supplementing knowledge about regions of heterochromatin and ribosomal DNA in this species, thus contributing to future elucidations about the genetic diversification in the genus Rogheessa.

alleni, R. gracilis, R. io and R. hussoni. The species occur exclusively in neotropical regions (Laval 1973;Genoways and Baker 1996;Ramiréz et al. 2014), and among them, R. minutilla, R. hussoni, and R. io are restricted to regions of South America (Gardner 2008). The Rhogeessa hussoni occurs in Suriname and Brazil and, in the latter, covers the states of Maranhão, Bahia (Gardner 2008). Recently had its distribution extended to the states of Sergipe (Mikalauskas et al. 2011), Mato Grosso, Minas Gerais, and Pará (Aires et al. 2011).
An explanation for this diverse karyotype within the genus Rhogeessa is the occurrence of chromosomal rearrangements by means of centric fusions and fissions (Baker et al. 1985;Baird et al. 2009).
Despite the efforts to acknowledge the variety of species of the genus Rhogeessa, the information about the R. hussoni and R. io occurring in the Brazilian territory is still scarce (Nogueira et al. 2014), with overlapping areas in the state of Mato Grosso (Gardner 2008;Aires et al. 2011). Rhogeessa hussoni and R. io are considered cryptic species, because of the difficult taxonomic designation through external morphological data (Gardner 2008;Gurgel-Filho et al. 2015). However, it is possible to distinguish the two species through their basic diploid number, being R. hussoni 2n=52 and R. io 2n=30 (Bickham and Baker 1977;Genoways and Baker 1996). The existing cytogenetic information about R. hussoni come from a specimen in the region of Suriname (Genoways and Baker 1996;Gardner 2008), and the authors presented only the basic karyotype of the species. However, there are no cytogenetic studies with mappings of specific chromosome regions for this species. In this study, we characterized the chromosome composition of a specimen of R. hussoni captured in Brazil, by identifying the sites of ribosomal DNA (rDNA) and the regions of constitutive heterochromatin (CBG bands) in this specimen's karyotype.

MATERIAL AND METHODS
The capture of a female specimen of Rhogeessa hussoni was made using mist nets (7.0 x 3.0) in the "Parque Municipal Mário Viana" Conservation Unit, located in the municipality of Nova Xavantina, state of Mato Grosso, Brazil. The area is characterized by the phytophysiognomy of Cerradão (14°42'02.6" S e 052° 21'01.5" W), inside the Brazilian Cerrado biome, with plant formations of continuous canopy and tree coverage ranging from 50% to 90% (Silva et al. 2008). The region's climate is tropical rainy (Aw) according to Köppen's classification system (Vianello and Alves 2012), with a dry season from April to September, and a rainy season from October to March (Pirani et al. 2009).
The female specimen of Rhogeessa hussoni was captured under the license 18276-1 from IBAMA/SISBIO/MT, and its identification was made based on specialized literature (Vizotto and Taddei 1973;Genoways and Baker 1996;Gardner 2008;Díaz et al. 2011). After the capture, the bat was kept in a cage until the following morning, when cytogenetic procedures were made. After extracting the biological material, the animal was mounted and deposited in the Scientific Collection of After the cytogenetic analyses, taxonomic characterizations were made to confirm species identification. External and cranial measurements were taken using a digital pachymeter (precision 0.01 mm) after the taxonomic characterizations. The measured features were: forearm length, total skull length, basal condyle length, canine condyle length, basal length, palatal length, length of upper teeth series, length of interior teeth series, mandible length, width of cingula (canine teeth), external width of molar teeth, interorbital width, postorbital width, zygomatic width, skull width, mastoid width, palatal width, skull height, and occipital height ( Fig. 1

and Supplementary
Material).

RESULTS
The morphometrical analyses confirmed that the exemplar was an R. hussoni.
We analyzed more than thirty metaphases to determine the diploid number (2N) and the number of autosomal arms (NF). The specimen of R. hussoni presented 2n = 52 and NF = 54 (Fig. 2). The set of autosomes is constituted by 23 pairs of acrocentric or subtelocentric chromosomes of large to small dimensions (pairs 1-12, 14-17, 19-25).
The sexual set is composed of two X chromosomes, medium size, with submetacentric morphology. The nucleolar organizing regions were evidenced in the pair of autosomal chromosomes number 25 (Fig. 2). With the C banding technique, we showed the formation of heterochromatin blocks in pericentromeric regions of all autosomal chromosomes. Regarding the sexual pair, one of the X chromosomes has a structure composed almost entirely by heterochromatin regions, whereas a small portion of heterochromatin is located in the pericentromeric region in the other X chromosome (Fig. 3a). In interphase nuclei, we observed the presence of regions of more condensed heterochromatin, which may suggest the inactivation of one of the X chromosomes ( Fig. 3b).

DISCUSSION
For species of the genus Rhogeessa, the sole use of morphological features does not allow a clear taxonomic designation, since Rhogessa hussoni and R. io show overlap in their forearm size (Gardner 2008). However, cytogenetic information provided fundamental data to designate species of the genus Baker 1996, Gardner 2008). The species Rhogeessa io and R. hussoni have geographical distribution in Brazilian territory with overlapping areas in the state of Mato Grosso (Gardner 2008;Gurgel-Filho et al. 2015).
The fist karyotype description of R. hussoni was of a specimen in Suriname (Genoways and Baker 1996), with 2n=52, whose authors assumed a metasubmetacentric morphology in sex chromosome X, which was followed in the present study. The morphology of the Y chromosome is not mentioned because there are still no cytogenetic descriptions of male specimens.
The karyotype of Myotis (2n=44) has been indicated as the similar ancestral karyotype for the family Vespertilionidae. Some species of the genus Eptesicus (2n=50, NF=48) are proposed as the closest evolutionary kinship of Rhogeessa (Bickham 1979).

Comparisons of G-bands patterns between chromosomes of Myotis velifer and
representatives of the genus Rhogeessa (R. parvula, R. túmida, R. aenus, and.R. io) showed homology between the autosomal chromosomes pairs 16/17 and 20/18, with rearrangements of chromosomal fusions shared by species of Rhogeessa presenting meta-submetacentric morphology (Bickham 1979;Baker et al. 1985). In the present study, the pair of chromosomes with meta-submetacentric morphology 13 of R. hussoni seems to correspond to pair 16/17 of Myotis and other species of Rhogeessa. Homology between G-bands patterns of pairs 20/18, which corresponds to the morphology of chromosome 18 found in R. hussoni, suggest a synapomorphy shared by species of the genus Rhogeessa (Bickham and Baker 1977;Baker et al. 1985).
Information about kinship relations has been obtained for some representatives of the family Vespertilionidae through comparative genomic techniques and G-bands patterns (Volleth et al. 2002(Volleth et al. , 2012Sotero-Caio et al. 2017). However, there is not a clear knowledge about chromosomal rearrangements in the genus Rhogeessa due to lacking data of cytogenetic bandings for some species.
The presence of a pair of ribosomal DNA sites was evidenced in R. hussoni.

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The blocks of heterochromatin in R. hussoni are ordered in pericentromeric regions in most autosomal chromosomes and in almost all the structure of one of the sex chromosomes X. (Fig. 3a). In Lasiurus ega, the presence of constitutive heterochromatin has also been evidenced along all the short arm of the X chromosome (Marchesin and Morielle-Versute 2004), in the same location of regions of heterochromatin in the sex chromosomes of R. hussoni. The organization of constitutive heterochromatin is dynamic among species. The existence of a copy of X chromosome, almost totally constituted by heterochromatin, with function in regulating the genic expression and gene dose compensation between complements XX and XY is acknowledged in several mammal species (Avner and Heard 2001). In R. hussoni, the presence of one X chromosome nearly wholly constituted by heterochromatin blocks, with regions of highly condensed interphase nuclei suggest a possible function in regulating gene expression of the X chromosome.
A large amount of information about cytogenetic and molecular comparisons for representative species of Myotis, Eptesicus, and Lasiurus is known (Bickham 1979;Varella-Garcia et al. 1989;Volleth et al. 2002Volleth et al. , 2012Larsen et al. 2012;Seim et al. 2013;Supanuam et al. 2012;Furman et al. 2014). Still, little is known about structural chromosome relations among the species of the genus Rhogeessa (Bickham 1979;Baker et al. 1985;Baker et al. 1992), and there are no studies about phylogenetic relationships based on molecular and cytogenetic markers for R. hussoni (Baird et al. 2009).
In the present study, we carried out the first cytogenetic characterization with chromosome bandings for R. hussoni, broadening knowledge about this species' chromosome composition. Further studies about ecological aspects, geographical distribution, molecular biology, and phylogenetic inferences are necessary to better understand and preserve the species, considering that the kinship relationship within the genus Rhogeessa is not clear. Besides, the species is classified as deficient in data from the International Union for Conservation of Nature (IUCN), reinforcing the importance of more data in order to better understand the status of ecological threat (Sampaio et al. 2016